Summary Telomeres, the DNA-protein complexes at the ends of chromosomes, serve apparently opposing functions: telomeres provide protective chromosome caps that ensure genome stability, yet telomeric DNA must also be accessible to interact with the chromosome end-replicating enzyme, telomerase. Recent work indicates that the human telomeric protein TPP1, which forms a heterodimer with POT1 and binds the single- stranded DNA extensions at the very ends of chromosomes, not only contributes to chromosome capping but also helps recruit telomerase and stimulates its processivity - the ability to add multiple telomeric DNA repeats after primer binding. The broad goal of the proposed research is to understand the molecular interactions responsible for telomerase activation by POT1-TPP1 and how much they contribute to telomere maintenance in vivo. Recently discovered separation-of-function mutants of TPP1, which retain full ability to form the POT1-TPP1-DNA complex but are defective in stimulating telomerase processivity, enable these experiments. Specific Aim 1 tests the hypothesis that a specific surface of TPP1 mediates telomerase processivity and determines whether the same surface is involved in telomerase recruitment in vitro. The approaches include direct assays of telomerase activity with the primer bound to POT1-TPP1 and separation-of-function mutants, and pull-down assays to assess binding. Specific Aim 2 addresses the interacting elements of the other partner, the telomerase itself. Exploiting sequence differences between the mouse and human telomerase RNAs, site-specific mutagenesis and activity assays will be used to identify RNA elements that contribute to POT1-TPP1 interaction. In addition, G100 on the surface of TERT is necessary for functional interaction with POT1-TPP1, and additional amino acid determinants will be identified. Specific Aim 3 addresses a key question: how much does the enhancement of telomerase activity by POT1-TPP1 seen in enzyme assays in vitro contribute to telomere maintenance in vivo? The separation- of-function TPP1 mutants will be integrated at single copy into an expression locus in a special HeLa cell line, the endogenous TPP1 will be knocked down using short hairpin RNAs, and telomere length, telomerase recruitment, and chromosome capping will be assessed. Specific Aim 4 addresses nonsynonymous SNPs (single nucleotide polymorphisms) of the hTERT gene that have been reported to be associated with diseases including dyskeratosis congenita, aplastic anemia, idiopathic pulmonary fibrosis, and cancer. The mutant TERTs will be assembled with telomerase RNA in human cells, immunopurified, and tested for defects in POT1-TPP1 stimulation of telomerase activity, which has the potential to reveal new disease mechanisms. The long-term goals of this work are to understand the mechanisms by which human telomeric DNA-binding proteins contribute to telomerase recruitment and telomeric repeat synthesis and to assess how mutations that perturb these processes contribute to human disease.